Compound for targeted degradation of BRD4 protein and preparation method and application thereof

Abstract

The invention discloses a compound for targeted degradation of BRD4 protein as well as a preparation method and application of the compound. Specifically, the invention discloses a complex shown as a formula I or a pharmaceutically acceptable salt thereof, or a racemic mixture, a hydrate, a solvate, a prodrug, an enantiomer, a diastereoisomer and a tautomer thereof, the structural formula of the complex is as follows: # imgabs0 #, in the formula, n is 1-10, R1 and R2 are # imgabs1 #, and X <-> represents an anion Cl <->. When the compound is used as a targeted BRD4 photodegradation agent, the space-time controllability and the biological safety of targeted protein degradation can be effectively enhanced. When the space-time controllability of illumination is utilized, the molecule can generate reactive oxygen substances at a fixed point of a tumor part under the irradiation of light. Compared with the traditional PROTAC molecule, the compound provided by the invention can play a role in specific time and spatial position more accurately, the biological safety is remarkably enhanced, and a new strategy and possibility are provided for the fields of tumor treatment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a compound for targeted degradation of BRD4 protein, and a preparation method and application thereof. Background Art

[0002] Cancer is a major global threat to human health. Currently, treatments for cancer primarily include surgery, radiotherapy, chemotherapy, and emerging immunotherapies. However, these approaches have limitations, such as significant side effects and the development of drug resistance. Therefore, the search for more effective and safe therapeutic targets has become a key research direction in cancer treatment. Bromodomain-binding domain protein 4 (BRD4), a key protein in epigenetic and transcriptional regulation, plays a crucial role in cellular physiology. It specifically recognizes acetylated lysine on chromatin and participates in the regulation of gene transcription. Studies have shown that dysregulated BRD4 expression is closely associated with the development of various cancers, including leukemia, breast cancer, and prostate cancer. Targeted inhibition of BRD4 activity can induce multiple cell death pathways, including apoptosis, autophagy, pyroptosis, and ferroptosis, effectively inhibiting tumor growth and spread. Therefore, BRD4 is considered a promising cancer therapeutic target and has garnered extensive attention and in-depth research from both the scientific community and the pharmaceutical industry in recent years, with ongoing drug development progress.

[0003] Proteolysis-targeting chimera (PROTAC) technology is an innovative targeted protein degradation technology that utilizes the ubiquitin-proteasome system in the cell to achieve degradation of specific target proteins. The traditional PROTAC molecule is a heterobifunctional small molecule whose structure consists of three parts: a target protein ligand, a linker, and an E3 ligase ligand. The target protein ligand can specifically bind to the target protein, the E3 ligase ligand is responsible for recruiting the E3 ligase, and the linker connects the two parts. When the PROTAC molecule enters the cell, it will bind to the target protein and the E3 ligase at the same time to form a target protein-PROTAC-E3 ligase ternary complex, thereby causing the target protein to be ubiquitinated and eventually degraded by the proteasome. The PROTAC molecule itself will be recycled after completing the degradation task and can continue to participate in the next round of degradation process. This "catalytic" degradation mechanism gives PROTAC technology the advantages of high efficiency and low toxicity.

[0004] PROTAC technology has shown tremendous potential in cancer treatment. By designing PROTAC molecules that target specific cancer-associated target proteins, it is possible to selectively degrade these target proteins, thereby achieving the goal of treating cancer. For example, designing PROTAC molecules targeting the BRD4 target protein is expected to provide a new and effective strategy for cancer treatment.

[0005] Currently, research on PROTAC molecules targeting BRD4 has made some progress, and some research results have been published in relevant academic journals. However, these PROTAC molecules have not yet entered the preclinical trial stage, mainly due to the following shortcomings of existing technologies:

[0006] 1. Uncontrollable mechanism of action: The process by which traditional PROTACs function is event-driven and uncontrollable. In practical applications, drug delivery systems can trigger off-target effects, where PROTACs bind to non-target proteins, causing unintended damage to normal tissues and cells. Such off-target effects not only reduce therapeutic efficacy but can also cause serious side effects, presenting a key challenge for the development of this field.

[0007] 2. Hook effect: When PROTAC concentration is high, a large number of binary complexes will form, namely PROTAC-target protein and PROTAC-E3 ligase binary complexes, rather than the expected target protein-PROTAC-E3 ligase ternary complexes. This hook effect can significantly affect the efficacy of PROTAC molecules, resulting in a decrease in their ability to degrade target proteins, limiting the application of PROTAC technology in cancer treatment.

[0008] 3. Design difficulties: As heterobifunctional small molecules, PROTAC molecules often do not meet the five principles of Lipinski drugs (i.e., molecular weight ≤ 500, number of hydrogen bond donors ≤ 5, number of hydrogen bond acceptors ≤ 10, logP ≤ 5, number of rotatable bonds ≤ 10). This increases the difficulty of PROTAC molecule design, making it difficult for researchers to develop PROTAC molecules with good pharmacokinetic properties and bioavailability, thus affecting the clinical translation of PROTAC technology.

[0009] In view of the shortcomings of the above-mentioned existing technologies, it is of great significance to develop a new PROTAC molecule with BRD4 as the target protein and its preparation method and application to solve the problems existing in the existing technologies, improve the efficacy and safety of PROTAC molecules and promote the clinical application of PROTAC technology in the field of cancer treatment. Summary of the Invention

[0010] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a compound that, when used as a photodegradation agent targeting BRD4, can effectively enhance the spatiotemporal controllability and biosafety of targeted protein degradation.

[0011] The present invention also provides a method for preparing the ruthenium complex.

[0012] The present invention also provides the application of the ruthenium complex.

[0013] According to one aspect of the present invention, a complex as shown in Formula I or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof is provided. The structural formula of the complex is as follows:

[0014]

[0015] Wherein, n is 1 to 10, R1 and R2 are X - Represents the anion Cl - .

[0016] The compounds according to the embodiments of the present invention have at least the following beneficial effects: The compound scheme proposed in the present invention can achieve photodynamic protein degradation function against BRD4. Compared with traditional PROTAC molecules targeting BRD4, the compounds of the present invention have several significant advantages: First, from the perspective of molecular structure and design and synthesis, the compounds of the present invention are monofunctional small molecules. Such monofunctional small molecules are structurally more likely to adhere to the five Lipinski principles of drug classifiers. The five Lipinski principles are important criteria for evaluating the oral bioavailability of drug molecules, and compliance with these principles means that the molecules have greater pharmacokinetic potential. In contrast, PROTAC molecules are mostly heterobifunctional structures, and their design and synthesis processes are much more complex than monofunctional small molecules, thus significantly reducing the design and synthesis difficulty of the compounds of the present invention. Second, the characteristics of monofunctional small molecules mean that they do not have the Hook effect that affects molecular function. The Hook effect generally refers to the phenomenon in some multivalent binding systems where high ligand concentrations lead to a decrease in binding efficiency. Because the compounds of the present invention are monofunctional small molecules, the negative effects of such complex interactions are avoided, thereby ensuring the stability and effectiveness of the molecular function. Third, in terms of biological activity, the compound molecule can specifically target and bind to the BRD4 protein in the cell. Under non-illumination conditions, it will not inhibit the function of BRD4, nor will it induce changes in the expression of BRD4. This characteristic enables the compound to maintain a relatively stable biological state when it is not subjected to specific stimulation, reducing interference with normal physiological processes. When the spatiotemporal controllability of light is utilized, the molecule can produce reactive oxygen species at the tumor site under light irradiation. Reactive oxygen species are highly oxidizing and can trigger a series of biochemical reactions, ultimately leading to the site-specific degradation of BRD4 protein. This light-based activation mechanism enables the compound molecules of the structure of the present invention to have a high degree of spatiotemporal controllability when exerting the target protein degradation function. Compared with traditional PROTAC molecules, the compounds of the present invention can act more accurately at specific time and spatial locations, significantly enhancing biosafety and providing new strategies and possibilities for fields such as tumor treatment.

[0017] According to some embodiments of the present invention, n is 6.

[0018] According to another aspect of the present invention, a method for preparing a complex as shown in Formula I is provided, comprising the following steps:

[0019] Reacting JQ-1 carboxylic acid with a compound represented by Formula II to produce intermediate I;

[0020] Ru 3+ React with 2,2-bipyridine to generate intermediate II, and then add 2,2-bipyridine-4-carboxyl to generate intermediate III;

[0021] reacting the intermediate I with the intermediate III to obtain a complex represented by formula I;

[0022] Wherein, the specific structures shown in Formula I and Formula II are as follows:

[0023]

[0024] Wherein, n is 1 to 10, R1 and R2 are X - Represents the anion Cl - .

[0025] The preparation method according to the embodiment of the present invention has at least the following beneficial effects: the preparation method of the present invention is simple to operate, the raw materials are cheap and easily available, the synthesis yield is high, and it has good production and application prospects.

[0026] According to some embodiments of the present invention, the Ru 3+ It is added in the form of a ruthenium salt or a hydrate thereof.

[0027] According to some embodiments of the present invention, the ruthenium salt is a halide salt, such as ruthenium chloride.

[0028] According to another aspect of the present invention, a photodegrader targeting BRD4 protein is also proposed. The raw materials for preparing the photodegrader include component A, and the component A includes the above-mentioned complex shown in Formula I or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof.

[0029] The photodegraders according to embodiments of the present invention have at least the following beneficial effects: the complexes described herein, or their pharmaceutically acceptable salt forms, as well as their racemic mixtures, hydrates, solvates, prodrug forms, enantiomers, diastereomers, and tautomers, all possess the ability to specifically bind to the BRD4 protein. These compounds not only target the BRD4 protein but also trigger its degradation under illumination. Current BRD4 protein degradation technologies primarily include PROTAC (Proteolysis Targeting Chimera) technology and combined photochemical degradation methods. Compared to existing technologies, the targeted BRD4 photodegraders of the present invention offer significant advantages: the ruthenium-based complex system can efficiently generate reactive oxygen species (ROS) under illumination, achieving targeted degradation of the intracellular BRD4 protein via a selective oxidative pathway. This mechanism, through precise spatial and temporal dual control (i.e., targeted binding and photoresponsive activation), effectively avoids the risk of nonspecific protein degradation potentially induced by traditional degraders, thereby significantly improving the safety and biocompatibility of targeted protein degradation drugs.

[0030] According to some embodiments of the present invention, the raw materials for preparing the photodegradation agent further include component B, and the component B includes gold nanoparticles (AuNPs).

[0031] The above-mentioned component A is combined with gold nanoparticles to prepare a complex, which can significantly increase the degradation rate of BRD4 protein under light.

[0032] According to some embodiments of the present invention, the BRD4 protein includes BRD4 in aqueous solution and / or BRD4 protein in cells.

[0033] According to another aspect of the present invention, the use of the complex shown in Formula I or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, tautomer thereof, or the photodegradant described above in the preparation of an anti-tumor drug is also provided.

[0034] According to the application of the embodiment of the present invention, there are at least the following beneficial effects: the present invention provides a photodynamic protein degradation system based on a ruthenium complex, specifically including a ruthenium complex and its pharmaceutically acceptable salts, stereoisomers (such as racemic mixtures, enantiomers, diastereomers), tautomers, hydrates, solvates and prodrug forms, or a photodegradation agent prepared based on the above complex, for achieving selective photodynamic degradation of BRD4 protein. The system designs a ruthenium complex or its derivatives as a targeted photosensitizer, and utilizes the short-range diffusion characteristics (migration distance of only a few nanometers) of reactive oxygen species (ROS, such as singlet oxygen 1O2 or hydroxyl radical ·OH) generated during photodynamic therapy (PDT) to achieve preferential damage to biomacromolecules in the vicinity of the photosensitizer. Combined with the specific recognition unit of the BRD4 protein (such as a polypeptide or small molecule inhibitor), the photosensitizer can accurately target the BRD4 protein, triggering its oxidative damage and conformational instability under light conditions, and then achieving protein degradation through the intracellular ubiquitin-proteasome system.

[0035] According to some embodiments of the invention, the tumor is non-small cell lung cancer.

[0036] According to some embodiments of the invention, the tumor is A549 non-small cell lung cancer.

[0037] According to some embodiments of the present invention, the tumor is a tumor caused by cisplatin-resistant A549R cells.

[0038] According to some embodiments of the present invention, the anti-tumor drug has anti-tumor activity under light conditions, and the light wavelength of the light conditions is 450±20 nm.

[0039] According to some embodiments of the present invention, the anti-tumor drug has anti-tumor activity under light conditions, and the wavelength of light under the light conditions is 450±10 nm.

[0040] According to some embodiments of the present invention, the anti-tumor drug has anti-tumor activity under light conditions, and the light wavelength of the light conditions is 450±5 nm.

[0041] According to some embodiments of the present invention, the illumination conditions include an illumination time of more than 5 minutes each time.

[0042] According to some embodiments of the present invention, the illumination conditions include an illumination time of more than 10 minutes each time.

[0043] According to some embodiments of the present invention, the illumination conditions include an illumination time of 5 to 15 minutes each time.

[0044] According to some embodiments of the present invention, the illumination condition includes an illumination intensity of 5 to 10 mW.

[0045] According to some embodiments of the present invention, the lighting condition includes a lighting intensity of 7.5 mW.

[0046] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the hydrogen spectrum of the intermediate product I prepared in the embodiment of the present invention.

[0048] Figure 2 This is the carbon spectrum of the intermediate product I prepared in the embodiment of the present invention.

[0049] Figure 3 The mass spectrum of the intermediate product I prepared in the embodiment of the present invention.

[0050] Figure 4 This is the hydrogen spectrum of the intermediate product III obtained in the embodiment of the present invention.

[0051] Figure 5 This is the carbon spectrum of the intermediate product III obtained in the embodiment of the present invention.

[0052] Figure 6 The mass spectrum of the intermediate product III obtained in the embodiment of the present invention.

[0053] Figure 7 This is the hydrogen spectrum of the complex prepared in the embodiment of the present invention.

[0054] Figure 8 This is the carbon spectrum of the complex prepared in the embodiment of the present invention.

[0055] Figure 9 The mass spectrum of the complex prepared in the embodiment of the present invention.

[0056] Figure 10 This is the UV-visible spectrum of the photodegradation agent RuIII@AuNPs prepared in an embodiment of the present invention.

[0057] Figure 11 This is the fluorescence spectrum of the photodegradation agent RuIII@AuNPs prepared in an embodiment of the present invention.

[0058] Figure 12 This is a signal diagram of TEMPO capturing singlet oxygen of the photodegradation agent RuIII@AuNPs prepared in an embodiment of the present invention.

[0059] Figure 13 This is an affinity diagram of the metal complexes prepared in the examples of the present invention.

[0060] Figure 14 This is a Western blot image of the cell thermal migration of the metal complex prepared in the example of the present invention.

[0061] Figure 15 This is a protein blotting image of the photodegradation agent RuIII@AuNPs prepared in an example of the present invention.

[0062] Figure 16 This is an immunofluorescence image of the BRD4 protein of the photodegradant RuIII@AuNPs prepared in an example of the present invention. DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. Unless otherwise specified, the test methods used in the embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be understood as limitations of the present invention.

[0064] In the description of the present invention, reference to the term "some embodiments" or the like indicates that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0065] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0066] The room temperature referred to in the present invention refers to 25±5°C, and is specifically 25°C in the embodiments of the present invention.

[0067] Example

[0068] This example provides a complex, the structural formula of which is shown in III below:

[0069]

[0070] The specific preparation process is as follows:

[0071] (1) JQ-1 carboxylic acid (59.6 mg, 0.15 mM), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 59.9 mg, 0.16 mM), and N,N-diisopropylethylamine (DIPEA, 24.5 mg, 0.36 mM) were added to N,N-dimethylformamide (1 mL). The mixture was stirred at room temperature for 1 h, and dichloromethane (3 mL) and trifluoroacetic acid (1 mL) were added. The mixture was stirred at room temperature for 4 h to achieve deprotection of the tert-butyloxycarbonyl group. Then, 50 mL of water was added. The mixture was extracted with dichloromethane. The volume of the extract was reduced by evaporation under reduced pressure. The crude product was purified using a 100-200 mesh silica gel column with dichloromethane / methanol (200:1, v / v) as the eluent to obtain intermediate I. Yield: 81%.

[0072] The synthesis process is as follows:

[0073]

[0074] Determine the hydrogen spectrum, carbon spectrum and mass spectrum of intermediate product I as follows Figures 1 to 3 shown.

[0075] 1 H NMR (400 MHz, deuterated methanol) δ 7.46–7.39 (m, 4H), 4.65–4.51 (m, 1H), 3.37 (s, 4H), 3.25–3.21 (m, 2H), 2.98 (s, 2H), 2.91–2.89 (m, 2H), 2.70 (s, 3H), 2.45 (s, 3H), 1.70 (s, 2H), 1.58 (d, J = 6.8 Hz, 2H), 1.38 (s, 10H). 13 C NMR (151 MHz, deuterated acetonitrile) δ 170.08, 164.24, 164.07, 162.50, 155.47, 150.59, 136.88, 136.13, 132.17, 131.60, 130.38, 130.24, 128.47, 54.07, 39.83, 38.87, 37.94, 37.90, 35.67, 29.13, 28.41, 26.81, 26.18, 25.66, 13.63, 11.01. ESI-MS: m / z = 527.33, ([M+H] + ).

[0076] It can be seen from the figure and the above test results that the target structure compound was obtained through the above operations.

[0077] (2) RuCl3·nH2O (1.56 g, 5.97 mM), 2,2-bipyridine (1.87 g, 11.97 mM), and LiCl (1.68 g, 39.63 mM) were dissolved in 10 mL of N,N-dimethylformamide, heated under reflux for 8 h under Ar protection, cooled to room temperature, added with 50 mL of acetone, and refrigerated at 4 ° C overnight to precipitate a solid. The solid was filtered, precipitated, and washed with ice water to obtain a dark black microcrystalline intermediate II. The solid was dissolved in a mixture of ethanol and water (V / V = 3:1). 2,2-bipyridine-4-carboxyl was added, refluxed at 80 ° C for 24 h, and cooled to room temperature. Then, the solvent was removed by rotary steam under reduced pressure. Purification was performed on an Al2O3 column to obtain a red solid intermediate III. Yield: 75%. The synthesis process is as follows:

[0078]

[0079] Determine the hydrogen spectrum, carbon spectrum and mass spectrum of intermediate III as follows Figures 4-6 shown.

[0080] 1 H NMR (400 MHz, deuterated methanol) δ 9.06 (d, J = 1.7 Hz, 1H), 8.79 (d, J = 8.2 Hz, 1H), 8.73 (d, J = 8.2 Hz, 4H), 8.14 (t, J = 6.9 Hz, 6H), 7.97 (d, J = 5.8 Hz, 1H), 7.90 (dd, J = 5.8, 1.7 Hz, 1H), 7.85–7.80 (m, 5H), 7.53–7.48 (m, 5H). 13 C NMR (151 MHz, deuterated methanol) δ 159.36, 158.60, 158.56, 158.54, 158.47, 158.43, 158.33, 153.36, 152.80, 152.74, 152.68, 152.65, 152.58, 139.49, 139.47, 139.45, 139.43, 139.32, 129.19, 129.08, 129.05, 129.00, 127.94, 126.03, 125.78, 125.74, 125.72, 124.73. Elemental analysis (%) calculated for C 31 H 24 Cl2N6O2Ru·6H2O:C:46.98,H:4.58,N:10.60.Found:C:46.84,H:4.22,N:10.70.ESI-MS:m / z=307.03([M-2Cl] 2+ ).

[0081] It can be seen from the figure and the above test results that the target structure compound was obtained through the above operations.

[0082] (3) Intermediate III (65.1 mg, 0.11 mM), intermediate I (52.2 mg, 0.098 mM), 4-dimethylaminopyridine (DMAP, 10.2 mg, 0.082 mM), 1-hydroxybenzotriazole (HOBt, 11.23 mg, 0.082 mM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC3·HCl, 15.5 mg, 0.082 mM), and N,N-diisopropylethylamine (DIAPE, 15.5 mg, 0.082 mM) were dissolved in N,N-dimethylformamide (2 mL) and stirred at room temperature for 14 h to obtain the complex (RuIII). Yield: 19%.

[0083] The synthesis process is as follows:

[0084]

[0085] Determine the hydrogen spectrum, carbon spectrum and mass spectrum of the complex Figures 7-9 shown.

[0086] 1 H NMR (400MHz, deuterated methanol) δ9.06 (d, J=2.4Hz, 1H), 8.72 (dd, J=8.5, 3.0Hz, 4H), 8.21–7 .95(m,6H),7.82(dt,J=11.8,5.9Hz,6H),7.59–7.44(m,6H),7.47–7.31(m,4H) ,4.62(dd,J=8.9,5.4Hz,1H),3.50(d,J=7.0Hz,4H),3.39(d,J=9.6Hz,2H),3.2 3–3.13(m,2H),2.69(s,3H),2.44(s,3H),1.67(d,J=8.3Hz,4H),1.37(s,10H). 13C NMR (151 MHz, deuterated methanol) δ 172.70, 166.25, 165.58, 161.32, 159.40, 158.55, 158.49, 158.43, 158.27, 157.09, 153.38, 152.79, 152.74, 152.60, 152.23, 144.25, 139.45, 138.17, 138.03, 133.59, 133.34, 132.05, 131.39, 129.82,129.29,129.07,126.30,126.10,125.78,125.75,123.15,57.93,55.32,45.16,41.44,40.50,38. 97,38.90,35.87,30.52,30.37,30.32,28.84,28.03,27.96,15.81,15.49,14.45,12.97,11.64.Elemental analysis(%)calcd.forC 58 H 57 Cl3N 12 O2RuS·6H2O:C:53.52,H:5.34,N:12.91,S:2.46.Found:C:53.42,H:5.54,N:12.77,S:2.35.ESI-MS:m / z=561.84([M-2Cl] 2+ ).

[0087] It can be seen from the figure and the above test results that the target structure compound was obtained through the above operations.

[0088] This example also produced a photodegradation agent, RuIII@AuNPs. Specifically, 2.6 mg of the RuIII complex prepared above was weighed and dissolved in 218 μL of DMSO to obtain a 10 mM stock solution. This solution was slowly added to 1 mL of a rapidly stirred gold nanoparticle solution, stirred at room temperature for 4 hours, and dialyzed to remove the free RuIII complex, forming RuIII@AuNPs (measured to have a particle size of approximately 2 nm and a zeta potential of +28.14 mV).

[0089] Test Case

[0090] (1) This example provides the UV absorption and fluorescence spectra of RuIII@AuNPs nanoparticles. The specific steps are as follows:

[0091] The RuIII@AuNPs nanoparticles obtained above were diluted 1000 times with ultrapure water, and their ultraviolet absorption was measured by an ultraviolet spectrophotometer, and their emission wavelength was measured by a fluorescence spectrophotometer. Figure 10 and 11 As shown in the figure, it can be seen that the maximum absorption of nanoparticles RuIII@AuNPs is 459nm and the emission wavelength is 621nm.

[0092] (2) This example also provides identification of the type of ROS produced by the metal complexes prepared in the examples of the present invention. The specific steps are as follows:

[0093] The measurements were performed at room temperature using an electron spin resonance spectrometer (Bruker A300) with tetramethylpiperazine oxide (TEMPO) as a singlet oxygen scavenger. The electron spin resonance spectrometer was tuned at 1G and scanned at 100G. The irradiation power of the RuIII@AuNPs nanoparticles was 20mW. TEMPO was used to spin-trap the singlet oxygen generated by the complex under illumination. The results are shown in Figure 2. Figure 12 As shown in Figure 3, compared with dark conditions, nanoparticles RuIII@AuNPs can produce singlet oxygen under light conditions.

[0094] (3) This example also provides an experiment to determine the affinity of the metal ruthenium complex RuIII for the BRD4 protein. The specific steps are as follows:

[0095] A 10 mM stock solution of the complex was diluted with PBS to a concentration of 500 μM. The concentrations were then diluted to 1.250, 0.625, 0.312, 0.156, 0.078, and 0.039 μM, respectively, using PBS solutions containing 5% DMSO and 0.5‰ P20 (polysorbate 20, also known as Tween 20; MedChemExpress, Cat. No. HY-141415). BRD4 protein was loaded onto a CM5 chip, and the binding ability of metal complex III to BRD4 protein was measured using a protein interaction platform (Biacore8k).

[0096] The results are as follows Figure 13 As shown in the figure, it can be seen that the Kd of RuIII and BDR4 obtained by the algorithm of the protein molecular interaction platform is 0.252 μM, which shows that the complex has a high affinity for BRD4.

[0097] (4) Furthermore, this example also conducted a cell thermal migration experiment of complex III, as follows:

[0098] Take 1×10 6A549R cells were seeded in 10 cm culture dishes for 2 days. The cells were digested with 0.25% trypsin, and total protein was extracted using a protein extraction kit. The extracts were then divided into two equal parts. 1 μM of complex III was added to one of the extracts and incubated for 30 minutes. Each extract was then divided into 6 parts and placed on a hot plate at different gradient temperatures (50, 53, 56, 59, 62, 65, 68°C) for 3 minutes and cooled at room temperature for 3 minutes. The supernatant was then centrifuged, heated at 95°C for 5 minutes to denature the protein, and analyzed by Western blotting.

[0099] The results are as follows Figure 14 As shown in the figure, it can be seen from the protein blotting results that in the group without RuIII addition, the degradation of BRD4 protein gradually accelerated with the increase of heating temperature, while in the group with RuIII addition, BRD4 protein can remain relatively stable during the heating process, indicating that RuIII can bind to BRD4.

[0100] (5) Protein blotting analysis of RuIII@AuNPs was also performed in this case, as follows:

[0101] 1×10 6 A549R cells were seeded in a 10 cm culture dish for one day and incubated with / without AuNPs (1 μg / mL), Ru1 (1 μM) and Ru1@AuNPs (1 μM) for 12 h. The cells were then irradiated with a 450 nm surface light source (7.5 mW, 10 min). After the irradiation, the cells were incubated for another 12 h, and then protein extraction was performed.

[0102] Protein extraction and denaturation: Lyse the sample using RIPA cell lysis buffer containing protease inhibitors and centrifuge to obtain the supernatant. Quantitative analysis using the BCA assay is performed on a portion of the supernatant. Add loading buffer to the remaining portion and boil for 5 minutes to completely denature the protein.

[0103] SDS-PAGE electrophoresis

[0104] Gel preparation: Prepare 8% of the required volume of gel according to the ratio of the SDS-PAGE gel preparation kit, mix well and add to the plywood, insert a 1.5mm 15-hole comb, and let it stand until the gel is formed.

[0105] Preparation of 1× electrophoresis buffer: Dissolve 3.0 g of Tris, 14.6 g of glycine, and 1.0 g of SDS in 1 L of deionized water.

[0106] Preparation of 1× transfer buffer: First add 5.8g of Tris and 2.9g of glycine to 800mL of deionized water, stir until completely dissolved, and then add 200mL of methanol.

[0107] Preparation of blocking solution: Dissolve 2.5 g of skim milk powder in 50 mL of TBS-T solution to make a 5% milk powder solution.

[0108] Sample loading and electrophoresis: Place the gel plate holder into the electrophoresis tank. Fill the inner tank with fresh 1x electrophoresis buffer. Add 2.5 μL of protein marker to each marker well and an equal amount of extracted protein to the remaining wells. Add 1x electrophoresis buffer to the outer tank, close the lid, and connect the power supply. Set the voltage to 60V for 30 minutes. Once the sample enters the separating gel, adjust the voltage to 100V. Terminate electrophoresis when the bromophenol blue indicator reaches the bottom of the gel (approximately 1 cm).

[0109] Transfer: Cut a PVDF membrane to a size that matches the gel / target protein location, place it in methanol, and activate it on a shaker for 30 seconds. Carefully remove the gel and cut the lower gel containing the target protein. Place a transfer sponge and filter paper on a plastic clip. Place the membrane in the order blackboard, sponge, filter paper, gel, PVDF membrane, filter paper, and sponge. Transfer current: 300 mA, transfer time: 60 minutes.

[0110] Blocking: Transfer the transferred membrane into the blocking solution and shake at room temperature for 1 hour.

[0111] Incubate with primary antibody: Transfer the blocked membrane to TBS-T solution for washing, then cut the membrane at 70 kDa, incubate with BRD4 protein antibody at 70 kDa, and incubate with internal control antibody Tubulin below 70 kDa, incubate overnight at 4°C.

[0112] Incubation with secondary antibody: After the membrane incubated with primary antibody was washed in TBS-T, the secondary antibody was added and incubated for another 1 hour at room temperature.

[0113] Development: SupersignalWestFemtoECL (ThermoScience TM Emm TM ) and visualized by OmegaLum CI Imaging System (Aplegen, USA).

[0114] The results are as follows Figure 15 As shown in the figure, it can be seen from the protein blot that compared with RuIII and AuNPs, gold nanoparticles RuIII@AuNPs can downregulate the expression of BRD4 protein under light conditions after entering the cells.

[0115] Furthermore, this example also conducted an immunofluorescence experiment of BRD4 protein of nanoparticles RuIII@AuNPs, as follows:

[0116] (1) Working fluid preparation:

[0117] Blocking solution: 500 μL fetal bovine serum + 9500 μL PBS, add 30 μL Triton-100 during shaking.

[0118] Antibody diluent: 30 μL Triton-100 + 10 mL PBS + 100 mg bovine serum albumin.

[0119] (II) Sample preparation:

[0120] 1×10 4 A549R cells were seeded in a 1.5 cm confocal culture dish and cultured for 1 day. AuNPs (1 μg / mL), RuIII (1 μM) and RuIII@AuNPs (1 μM) were added / not added, and then incubated for 12 h. The cells were then illuminated (7.5 mW, 10 min) / darkly treated with a 450 nm surface light source, and incubated for another 12 h after the illumination ended.

[0121] (III) Fixation:

[0122] After incubation, the medium was discarded, the cells were washed once with PBS, 600 μL of paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 min. The cells were then washed three times with PBS for 5 min each time.

[0123] (IV) Closure:

[0124] Add 600 μL of blocking solution to each well and incubate at room temperature for 1 h.

[0125] (V) Antibody incubation:

[0126] The blocking solution was discarded, and BRD4 antibody was diluted with antibody diluent at a ratio of 100:1, 60 μL was added to each well, and incubated at 4°C overnight.

[0127] (VI) Fluorescent secondary antibody incubation:

[0128] Discard the antibody incubation solution and wash three times with PBS, each for 5 minutes. Dilute the AlexaFluor 555 fluorescent secondary antibody 1000:1 in antibody diluent, add 100 μL to each well, and incubate at room temperature for 1.5 hours. Wash three times with PBS, each for 5 minutes.

[0129] (VII) Imaging: Imaging was performed using a confocal microscope.

[0130] The results are as follows Figure 16As shown in the figure, it can be seen that under light conditions, after the addition of RuIII and RuIII@AuNPs, the fluorescence intensity in the cells was significantly weakened, especially the RuIII@AuNPs group was further significantly weakened compared with RuIII, indicating that RuIII@AuNPs significantly increased the degradation rate of BRD4 protein in the cells under light conditions; this also shows that RuIII@AuNPs further increased the uptake of complex III by the nucleus of A549R cells.

[0131] The results of biological application experiments in which the complex of the present invention targets BRD4 in cells and performs photodegradation show that the complex of the present invention can generate reactive oxygen species at both the solution and cellular levels under light conditions. In addition, the Kd of the complex and BRD4 protein was obtained by experiment = 0.252 μM. Furthermore, after incubating tumor cells for 12 hours, the complex was illuminated with a 450nm surface light source (7.5mW, 10min), and a large amount of ROS was generated in the cells, and the BRD4 protein content in the cells was significantly downregulated, which clearly indicates that the complex of the present invention can target BRD4 in light-degrading cells through ROS generated by light.

[0132] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. A complex as represented by formula I or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof, characterized in that: The structural formula of the complex is as follows: Wherein, n is 1 to 10, R1 and R2 are X - Represents the anion Cl - .

2. The complex according to claim 1 or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof, characterized in that: Said n is 6; and / or, X - Represents Cl - .

3. A method for preparing the complex as shown in formula I, characterized in that: The steps include: Reacting JQ-1 carboxylic acid with a compound represented by Formula II to produce intermediate I; Ru 3+ React with 2,2-bipyridine to generate intermediate II, and then add 2,2-bipyridine-4-carboxyl to generate intermediate III; reacting the intermediate I with the intermediate III to obtain a complex represented by formula I; Wherein, the specific structures shown in Formula I and Formula II are as follows: Wherein, n is 1 to 10, R1 and R2 are X - Represents the anion Cl - .

4. A photodegrader targeting BRD4 protein, characterized by: The raw materials for preparing the photodegradant include component A, which includes the complex as described in claim 1 or 2 or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof.

5. The photodegradation agent targeting BRD4 protein according to claim 4, characterized in that: The raw materials for preparing the photodegradant further include component B, and the component B includes nano-gold particles.

6. Use of the complex according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, or a racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof, or the photodegradant according to claim 4 or 5 in the preparation of an antitumor drug.

7. The use according to claim 6, characterized in that: The tumor is non-small cell lung cancer; preferably, the tumor is A549 non-small cell lung cancer.

8. The use according to claim 6 or 7, characterized in that: The anti-tumor drug has anti-tumor activity under light conditions, and the light wavelength of the light conditions is 450±20nm.

9. The use according to claim 8, characterized in that: The illumination conditions include an illumination time of 5 to 15 minutes each time.

10. The use according to claim 8, characterized in that: The illumination conditions include an illumination intensity of 5 to 10 mW.

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